The elucidation of the molecular mechanisms of the regulation of cellular proliferation is one of the fundamental goals in cell biology. Sphingosine, a metabolite of membrane sphingolipids, may play an important role in the regulation of cell growth, yet our knowledge in this area is immature and fragmentary. Our preliminary observation that sphingosine stimulates the growth of quiescent cultures of Swiss 3T3 fibroblasts raises the intriguing possibility that these breakdown products of cellular sphingolipids may function as modulators of cellular proliferation and possibly as a new class of lipid second messengers. To elucidate the mechanisms by which sphingosine induces cellular proliferation, its effects on several signaling systems which play a vital role in cell growth regulation will be examined. Initially, emphasis will be on its effect on protein kinase C, a key regulatory enzyme in signal transduction during cellular proliferation, as it has been shown that sphingosine is an inhibitor of protein kinase C in vitro and of those processes dependent on this enzyme. Our preliminary results suggest that sphingosine may mediate its mitogenic effect by a fundamentally different, protein kinase C- independent pathway. This point has to be firmly established as it has important implications for the use of sphingosine as a protein kinase C inhibitor in vivo and for the existence of other targets for its action. To extend the studies to other targets, the effect of sphingosine on known intracellular second messengers including, cAMP, intracellular free Ca+2, diacyl glycerol, and phosphoinositides will be thoroughly investigated. Since it is well established that the activation of multiple protein kinases plays a prominent role in the regulation of cell growth, alterations in the pattern of cellular phosphorylation by mitogenic concentrations of sphingosine will be determined. A search for a novel sphingosine-modulated protein kinase will then be initiated. A critical test of the second messenger hypothesis will be the measurement of changes in endogenous sphingosine levels. Comparison of basal sphingosine levels, turnover, and de novo synthesis in the various cell types might yield valuable information regarding the role of sphingosine in cell proliferation. Studies on other targets for the actions of sphingosine will be of great importance in elucidating its physiological significance and potential use as a pharmacological agent. Determination of the underlying molecular mechanisms of the effect of sphingosine might open a window for better understanding of this new class of second messengers. Furthermore, these studies may reveal important clues to the pathophysiology of the sphingolipidoses disorders.